Diffusion MRI has the potential to probe the compartmental origins of MR signals acquired from human nervous tissue. However, current experiments in human subjects require long diffusion times, which may confound data interpretation due to the effects of compartmental exchange. To investigate human nervous tissue at shorter diffusion times, and to determine the relevance of previous diffusion studies in rat hippocampal slices, water diffusion in 20 perfused human hippocampal slices was measured using a wide-bore 17.6-T magnet equipped with 1000-mT/m gradients. These slices were procured from five patients undergoing temporal lobectomy for epilepsy. Tissue viability was confirmed with electrophysiological measurements. Diffusion-weighted water signal attenuation in the slices was well-described by a biexponential function (R 2 > 0.99). The mean diffusion parameters for slices before osmotic perturbation were 0.686 ؎ 0.082 for the fraction of fast diffusing water (F fast ), 1.22 ؎ 0.22 ؋ 10 -3 mm 2 /s for the fast apparent diffusion coefficient (ADC), and 0.06 ؎ 0.02 ؋ 10 -3 mm 2 /s for the slow ADC. Slice perturbations with 20% hypotonic and 20% hypertonic artificial cerebrospinal fluid led to changes in F fast of -8.2% and ؉10.1%, respectively (ANOVA, P < 0.001). These data agree with previous diffusion studies of rat brain slices and human brain in vivo, and should aid the development of working models of water diffusion in nervous tissue, and thus increase the clinical utility of diffusion MRI.

Objectives Congenital heart disease (CHD) is a common disease of human birth defects, and atrial septal defect (ASD) is the most common form of ASD. Genetic variants in the coding region of the CITED2 gene is known to be significantly correlated with cardiac malformations, but variants of the CITED2 promoter region and its relationship with the formation of ASD are still unclear. We hypothesize that the variants of the CITED2 promoter may be related to pathogenesis of ASD. The purpose of this study was to screen variants in the promoter region of the CITED2 gene and to verify the effect of the variants on gene expression at the cellular level. Methods The blood samples of 332 ASD patients and 293 unrelated healthy children used as controls were studied. The total DNA of all subjects was extracted, and the CITED2 promoter variants were screened by PCR combined with Sanger sequencing. The luciferase activity of the CITED2 promoter was measured by a dual luciferase reporter at the cellular level. Electrophoretic mobility change assay (EMSA) and bioinformatics analysis were used to test the effect of CITED2 promoter changes on transcription factor binding sites. Results Four variants in the promoter region of the CITED2 gene were found only in the 332 ASD patients with zero occurrence in the 293 control subjects. These included one novel heterozygous variant (g.4933C>A) and three SNPs [1 case of g.4078 A>C(rs1165649373), 4 cases of g.4935C>T(rs111470468), 2 cases of g.5027C>T (rs112831934)]. The cellular functional analysis showed that these four variants significantly changed the transcriptional activity of the CITED2 gene promoter in HEK‐293 cells (P<0.05). In addition, EMSA results and database analysis showed that these variants created or destroyed a series of possible transcription factor binding sites, resulting in changes of the expression of CITED2 protein. Conclusion The variants of CITED2 promoter sequence in ASD patients affect transcriptional activity and are likely involved in the occurrence and development of ASD. These findings may provide new perspectives on the molecular pathogenesis and potential therapeutic insights of ASD patients.

ObjectiveCongenital heart defects (CHD) represent the most common congenital anomaly in newborns. The causes of CHD are complex, and are not fully understood. A number of genetic studies have linked gene mutations to cardiac abnormalities. In this study, we found a child who was diagnosed as having a complex including complete endocardial cushion defect, patent ductus arteriosus, secondary atrial septal defect, severe pulmonary hypertension, and polydactyly, we aimed to identify potential pathogenic mutations from this complex.MethodsThe whole blood was collected and genomic DNA was extracted to identify mutations by whole exome sequencing (WES). The CRISPR/Cas9 system was used to generate human pluripotent stem cell with mutations (hPSC‐Mut) separately. As pluripotency markers, Oct4, Nanog, Klf4, SSEA4 were tested in hPSC‐Mut cell, by the methods of Immunofluorescence, Real‐Time PCR, Western Blot. The hPSC‐Mut cell was then inducted and differentiated into cardiomyocytes (CM‐Mut), The differentiation efficiency and contraction of CM‐Mut was detected in 0d, 2d, 4d, 8d, 13d.ResultTwo heterozygous mutations, LTBP2 (c.2206G>A), TCTN3 (c.1268G>A) were identified via WES and analysed by bioinformatics. The hPSC‐LT/TC stable cell line was constructed, inducted and differentiated into CM‐LT/TC. Compare to the wild type, there were no significant differences in cell pluripotency and differentiation efficiency. The cell contraction was observed in the 8th day and lasted to the 13rd day, the contraction of CM‐LT was faster and CM‐LT was slower than wild type cell line.ConclusionTwo heterozygous mutations LTBP2 (c.2206G>A) and CTN3 (c.1268G>A) with alteration of the contraction of CM‐LT/LT cell were found to be pathogenic in complex CHD.Support or Funding InformationNational Natural Science Foundation of China (81870288, 81641017), National Central Grants for Research Institute (2017NL31001), Binhai New Area Health Bureau ( 2016BWKZ 003).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

BackgroundEndogenous H2S generated by mercaptopyruvate sulfurtransferase (MPST) coupled with cysteine aminotransferase (CAT) in endothelium as a physical vasodilator and blood pressure regulator may play an important protective role in the pathogenesis of coronary heart disease. The present study aimed to investigate the role of cGMP and endothelium‐derived hyperpolarizing factor (EDHF) in H2S‐induced vasorelaxation in the human internal mammary artery (IMA).MethodsIn the human IMA segments from 54 patients undergoing coronary artery bypass grafting, the acetylcholine (ACh)‐induced relaxation and the effect of CAT inhibitor (aminooxyacetic acid [AOAA] and L‐aspartate) and KCa channel blocker or KATP channel blocker were studied in a myograph. Enzyme‐linked Immunosorbent Assay for PDE5A and western blotting analysis for eNOS and p‐eNOSser1177 were performed. Methylene blue assay was used to quantify the contents of H2S.ResultsThe maximal relaxation induced by ACh was significantly attenuated by AOAA or L‐aspartate, Iberiotixin (BKCa channel blocker) (P<0.01), or IKCa channel blocker plus SKCa channel blocker (TRAM‐34+Apamin) (P<0.01), or KATP channel blocker (Glibenclamide) (P<0.01). The H2S donor NaHS increased eNOS phosphorylation at its activating site S1177 and down‐regulated PDE5A activity. Incubation with ACh for 4 hours significantly increased the H2S concentration.ConclusionsWe for the first time demonstrated in the human IMA that endogenous H2S could be produced under the stimulation of ACh and that CAT is one of the key enzymes in production of H2S in the endothelial cell of the IMA. H2S may activate eNOS to produce NO that activates PKG and may also inhibit PDE5A activity; both enhance the cGMP pathway‐mediated relaxation. Further, H2S as a potential EDHF directly stimulates KCa channels to induce vasodilatation. These findings may provide new therapeutical targets for the treatment of vasospasm of coronary artery bypass grafting vessels and provide a pharmacological basis for the development of new vasodilator drugs.Support or Funding InformationSupported by grants from the National Natural Science Foundation of China (81170148 & 81641017), Zhejiang Provincial Natural Science Foundation (LY15H020008), & Tianjin Binhai Key Platform for Creative Research Program (2012‐BK110004), Binhai New Area Health Bureau (2012BWKZ008, 2016BWKY007, 2016BWKZ003).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.